Glaucoma is an optic nerve disorder characterized by cupping of the optic nerve head and loss of peripheral vision. Occasionally there is also loss of central vision. In the majority of patients, an elevated intraocular pressure is present and is thought to contribute to the optic nerve damage. Glaucoma is the second leading cause of blindness in developed countries (Leske, M. C. (1983) Am. J. of Epidemiology 118:166-191). Its prevalence increases with age and is greater in black patients (Leske, M. C. (1983) Am. J of Epidemiology 118:166-191). Glaucoma affects approximately 2.3 million Americans and blinds approximately 12,000 of them per year (Tielsch, J. M. (1993) Therapy for glaucoma: costs and consequences. In Transactions of the New Orleans Academy of Ophthalmologists, S. F. Ball, Franklin, R. M. (Ed.), pp 61-68. Kugler, Amsterdam).
The most prevalent form of glaucoma is primary open angle glaucoma (POAG), a progressive disease of the optic nerve characterized by degeneration and cupping of the optic nerve, loss of peripheral visual field, and increased intra-ocular pressure. Evidence indicates that POAG is genetically heterogeneous with a complex mode of inheritance. An early onset form of POAG known as juvenile open angle glaucoma (JOAG) is an autosomal dominant disorder with high penetrance.
A significant fraction of glaucoma has a genetic basis (Benedict, T. W. G. Abhaundlungen zus dem Gebiete der Augenheilkunde. Breslau: L. Freunde (1842); Stokes, (1940) W. Arch Ophthalmol 24:885-909; Kellerman, L. and A. Posner, (1955) Am. J. Ophthalmol.; 40:681-685; Becker, B., et al., (1960) Am. J. Ophthalmol. 50:557-567; Francois, J., et. al., (1966) Am. J. Ophthalmol.; 62:1067-1071; Armaly, M. F. (1967) Arch Ophthalmol; 78:35-43; Davies, T. G. (1968) Br. J. Ophthalmol.: 52:31-39; Jay, B., Paterson, G. (1970) Trans. Ophthalmol. Soc. U.K.; 90:161-171; Paterson, G. (1970) Trans. Ophthalmol. Soc. U.K.; 90:515-525; Miller, S.J.H. (1978) Trans. Ophthalmol. Soc. U.K. 98:290-292), which allows genetic methods to be used to investigate the pathophysiological mechanisms of the disease at the molecular level. The chromosomal locations of genes causing three genetically distinct types of primary open angle glaucoma have been identified (Sheffield, V., et al. (1993) Nature Genetics 4:47-50; Sunden, S. L. F., et al. (1996) 6:862-869; Richards, J. E., et al. (1994) Am. J. Hum. Genet.: 54:62-70; Wiggs, J. L., et al. (1994) Genomics; 21:299-303; Stoilova, D., et al. (1996) Genomics 36:142-150; Wirtz, M. K., et al. (1997) Am. J. Hum. Genet. 60:296-304).
Therapeutics, which modulate (agonize or antagonize) genes (wild-type or mutant) involved in glaucoma, would be useful for the prevention and treatment of glaucoma. In addition, the detection of mutations in genes that correlate with the existence or a predisposition to the development of glaucoma can provide useful diagnostics.
In one aspect, the invention features isolated GLC1A nucleic acid molecules. The disclosed molecules can be non-coding, (e.g. probe, antisense or ribozyme molecules) or can encode a functional polypeptide (e.g. a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one bioactivity of a myocilin polypeptide).
In further embodiments, the nucleic acid molecule is a GLC1A nucleic acid that is at least 70%, preferably 80%, more preferably 85%, and even more preferably at least 95% homologous in sequence to the nucleic acids shown as SEQ ID No. 7 or 9 or to the complement thereof In another embodiment, the nucleic acid molecule encodes a polypeptide that is at least 92% and more preferably at least 95% similar in sequence to the polypeptide shown in SEQ ID No: 8 or 10.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least about 6 consecutive nucleotides of the sequences set forth as SEQ ID Nos: 1, 2, 3, 4, 5 or 6 or complements of the sequences set forth as SEQ ID Nos: 1, 2, 3, 4, 5 or 6 or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject GLC1A nucleic acids can include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer or suppressor sequence, which regulatory sequence is operably linked to the GLC1A gene sequence. Such regulatory sequences in conjunction with a GLC1A nucleic acid molecule can provide a useful vector for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing GLC1A proteins by employing said expression vectors.
In another aspect, the invention features isolated myocilin polypeptides, preferably substantially pure preparations, e.g. of plasma purified or recombinantly produced myocilin polypeptides. In one embodiment, the polypeptide is identical to or similar to a myocilin protein represented in SEQ ID No: 8 or 10. Related members of the vertebrate and particularly the mammalian myocilin family are also within the scope of the invention. Preferably, a myocilin polypeptide has an amino acid sequence at least about 92% homologous and preferably at least about 95%, 96%, 97%, 98% or 99% homologous to the polypeptide represented in SEQ ID No: 8 or 10. In a preferred embodiment, the myocilin polypeptide is encoded by a nucleic acid which hybridizes with a nucleic acid sequence represented in one of SEQ ID No: 7 or 9. The subject myocilin proteins also include modified proteins, which are resistant to post-translational modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The myocilin polypeptide can comprise a full length protein, such as represented in SEQ ID No: 8 or 10, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 460, 470, 475, 480, 485, or 490 amino acids in length.
Another aspect of the invention features chimeric molecules (e.g. fusion proteins) comprised of a myocilin protein. For instance, the myocilin protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the myocilin polypeptide (e.g. the second polypeptide portion is glutathione-S-transferase, an enzymatic activity such as alkaline phosphatase or an epitope tag).
Yet another aspect of the present invention concerns an immunogen comprising a myocilin polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a myocilin polypeptide; e.g. a humoral response, an antibody response and/or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from the protein represented in SEQ ID Nos: 8 or 10.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the myocilin protein. In preferred embodiments the antibody specifically binds to at least one epitope represented in SEQ ID Nos: 8 or 10.
The invention also features transgenic non-human animals which include (and preferably express) a heterologous form of a GLC1A gene described herein, or which misexpress an endogenous GLC1A gene (e.g., an animal in which expression of one or more of the subject GLC1A proteins is disrupted). Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed GLC1A alleles or for use in drug screening. Alternatively, such a transgenic animal can be useful for expressing recombinant myocilin polypeptides.
In yet another aspect, the invention provides assays, e.g., for screening test compounds to identify inhibitors, or alternatively, potentiators, of an interaction between a myocilin protein and, for example, a virus, an extracellular ligand of the myocilin protein, or an intracellular protein which binds to the myocilin protein.
A further aspect of the present invention provides a method of determining if a subject is at risk for glaucoma or another disorder resulting from a mutant GLC1A gene. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a myocilin protein, (e.g., a gene represented in one of SEQ ID Nos: 7 or 9, or a homolog thereof or a mutation of a GLC1A intronic sequence, e.g. as represented in SEQ ID Nos. 1-6); or (ii) the mis-expression of a GLC1A gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a GLC1A gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene (e.g., due to a promoter mutation); the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble myocilin protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer comprised of an oligonucleotide which hybridizes to a sense or antisense sequence of a GLC1A gene or naturally occurring mutants thereof, or intronic flanking sequences naturally associated with the GLC1A gene; (ii) contacting the probe/primer to an appropriate nucleic acid containing sample; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the GLC1A gene and, optionally, of the flanking nucleic acid sequences. For instance, the primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a GLC1A protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the myocilin protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.